Luciferase-based Reporter Gene Assays Research Articles

Naturally occurring small molecules with dual effect upon inflammatory signaling pathways and endoplasmic reticulum stress response

The endoplasmic reticulum (ER) is determinant to maintain cellular proteostasis. Upon unresolved ER stress, this organelle activates the unfolded protein response (UPR). Sustained UPR activates is known to occur in inflammatory processes, deeming the ER a potential molecular target for the treatment of inflammation. This work characterizes the inflammatory/UPR-related molecular machinery modulated by an in-house library of natural products, aiming to pave the way for the development of new selective drugs that act upon the ER to counter inflammation-related chronic diseases. Starting from a library of 134 compounds of natural occurrence, mostly occurring in medicinal plants, nontoxic molecules were screened for their inhibitory capacity against LPS-induced nuclear factor kappa B (NF-κB) activation in a luciferase-based reporter gene assay. Since several natural products inhibited NF-κB expression in THP-1 macrophages, their effect on reactive oxygen species (ROS) production and inflammasome activation was assessed, as well as their transcriptional outcome regarding ER stress. The bioactivities of several natural products are described herein for the first time. We report the anti-inflammatory potential of guaiazulene and describe 5-deoxykaempferol as a novel inhibitor of inflammasome activation. Furthermore, we describe the dual potential of 5-deoxykaempferol, berberine, guaiazulene, luteolin-4’-O-glucoside, myricetin, quercetagetin and sennoside B to modulate inflammatory signaling ER stress. Our results show that natural products are promising molecules for the discovery and pharmaceutical development of chemical entities able to modulate the inflammatory response, as well as proteostasis and the UPR.

Hsa_circ_0001658 accelerates the progression of colorectal cancer through miR-590-5p/METTL3 regulatory axis.

As reported, multiple circular RNAs (circRNAs) interfere with colorectal cancer (CRC) progression. Here, circRNA_0001658 (circ_0001658) is focused on studying how it works in CRC. Clarify the expression pattern, biological function, and underlying mechanism of circ_0001658 of CRC tumorigenesis. In CRC-related chip data retrieved using the database named Gene Expression Omnibus, different expressions of circRNAs between CRC and normal tissue samples were identified. Quantitative Real-time PCR and Western blot ensured the analysis on circ_0001658, microRNA-590-5P (miR-590-5p), and methyltransferase-like 3 (METTL3) mRNA expressions in tissues and cells. Cell counting kit-8 and flow cytometry were used to detect cell proliferation, apoptosis and migration. The targeting relations between circ_0001658, miR-590-5p, and METTL3 mRNA 3'-untranslated region were under the verification of bioinformatics prediction and dual luciferase-based reporter gene assays. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were employed on the downstream targets of miR-590-5p using the Database for Annotation, Visualization and Integrated Discovery database. Circ_0001658 and METTL3 mRNA was elevated in CRC tissues and cells, whereas miR-590-5p was decreased. Circ_0001658 overexpression promoted the proliferation of HT29 cells, inhibited apoptosis, and accelerated the cell cycle. In SW480 cells, knocking down circ_0001658 had the opposite effect. Circ_0001658 could specifically bind to miR-590-5p and negatively modulate its expressions; METTL3 is a miR-590-5p target that can be positively regulated by circ 0001658. Circ 0001658 was inversely associated with miR-590-5p expression while positively with METTL3 expressions. Circ_0001658 regulates the miR-590-5p/METTL 3-axis to increase CRC cell growth and decrease apoptosis.

A Setmelanotide-like Effect at MC4R Is Achieved by MC4R Dimer Separation.

Melanocortin 4 receptor (MC4R) is part of the leptin-melanocortin pathway and plays an essential role in mediating energy homeostasis. Mutations in the MC4R are the most frequent monogenic cause for obesity. Due to increasing numbers of people with excess body weight, the MC4R has become a target of interest in the search of treatment options. We have previously reported that the MC4R forms homodimers, affecting receptor Gs signaling properties. Recent studies introducing setmelanotide, a novel synthetic MC4R agonist, suggest a predominant role of the Gq/11 pathway regarding weight regulation. In this study, we analyzed effects of inhibiting homodimerization on Gq/11 signaling using previously reported MC4R/CB1R chimeras. NanoBRETTM studies to determine protein–protein interaction were conducted, confirming decreased homodimerization capacities of chimeric receptors in HEK293 cells. Gq/11 signaling of chimeric receptors was analyzed using luciferase-based reporter gene (NFAT) assays. Results demonstrate an improvement of alpha-MSH-induced NFAT signaling of chimeras, reaching the level of setmelanotide signaling at wild-type MC4R (MC4R-WT). In summary, our study shows that inhibiting homodimerization has a setmelanotide-like effect on Gq/11 signaling, with chimeric receptors presenting increased potency compared to MC4R-WT. These findings indicate the potential of inhibiting MC4R homodimerization as a therapeutic target to treat obesity.

Novel assays monitoring direct glucocorticoid receptor protein activity exhibit high predictive power for ligand activity on endogenous gene targets.

Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and auto-immune diseases. Unfortunately, their use is hampered by many side effects and therapy resistance. Efforts to find more selective glucocorticoid receptor (GR) agonists and modulators (called SEGRAMs) that are able to separate anti-inflammatory effects via gene repression from metabolic effects via gene activation, have been unsuccessful so far. In this study, we characterized a set of functionally diverse GR ligands in A549 cells, first using a panel of luciferase-based reporter gene assays evaluating GR-driven gene activation and gene repression. We expanded this minimal assay set with novel luciferase-based read-outs monitoring GR protein levels, GR dimerization and GR Serine 211 (Ser211) phosphorylation status and compared their outcomes with compound effects on the mRNA levels of known GR target genes in A549 cells and primary hepatocytes. We found that luciferase reporters evaluating GR-driven gene activation and gene repression were not always reliable predictors for effects on endogenous target genes. Remarkably, our novel assay monitoring GR Ser211 phosphorylation levels proved to be the most reliable predictor for compound effects on almost all tested endogenous GR targets, both driven by gene activation and repression. The integration of this novel assay in existing screening platforms running both in academia and industry may therefore boost chances to find novel GR ligands with an actual improved therapeutic benefit.

KDM5B promotes cell migration by regulating the noncanonical Wnt/PCP pathway in Hirschsprung’s disease

We measured the expression of the histone demethylase lysine-specific demethylase 5B (KDM5B) in the bowels of patients with Hirschsprung's disease (HSCR) and investigated the molecular mechanism by which KDM5B promotes the migration of neuronal PC12 cells. KDM5B expression was detected in the ganglionic and aganglionic colon of patients with HSCR (n = 10) and controls (n = 10). The expression and localization of KDM5B were assessed using immunohistochemical and immunofluorescence staining. Real-time PCR and Western blotting were performed to quantify KDM5B expression. The migration was determined using Transwell and wound-healing assays. G-LISA, GTPase pulldown and luciferase-based reporter gene assays were performed to evaluate the key components of Wnt/planar cell polarity (PCP) signaling in vitro. Our current study showed that KDM5B colocalized with neurons. KDM5B expression was reduced in HSCR specimens, while the aganglionic segments showed the greatest reduction. KDM5B knockdown inhibited the migration of PC12 cells. Moreover, inhibition of KDM5B decreased the expression of key genes in the Wnt/PCP pathway, and its inhibitory effect on PC12 cell migration was reversed by Wnt5a treatment. KDM5B promotes neuronal migration via the Wnt/PCP pathway. A potential role for KDM5B in altered enteric nervous system development in HSCR warrants further investigation.

Design and Characterization of a Fluorescent Reporter Enabling Live-cell Monitoring of MCT8 Expression.

Development and characterization of fluorescent MCT8 reporters that would permit live-cell monitoring of MCT8 protein expression in vitro in human induced pluripotent stem cell (hiPSC)-derived cell culture models. A tetracysteine (TC) motif was introduced into the human MCT8 sequence at four different positions as binding sites for fluorescent biarsenical dyes. Human Embryonic Kidney 293 cells were transfected and stained with fluorescein-arsenical hairpin-binder (FlAsH). Counterstaining with specific MCT8 antibody was performed. Triiodothyronine (T3) uptake was indirectly measured with a T3 responsive luciferase-based reporter gene assay in Madin-Darby Canine Kidney 1 cells for functional characterization. FlAsH staining and antibody counterstaining of all four constructs showed cell membrane expression of all MCT8 constructs. The construct with the tag after the first start codon demonstrated comparable T3 uptake to the MCT8 wildtype. Our data indicate that introduction of a TC-tag directly after the first start codon generates a MCT8 reporter with suitable characteristics for live-cell monitoring of MCT8 expression. One promising future application will be generation of stable hiPSC MCT8 reporter lines to characterize MCT8 expression patterns during in vitro neuronal development.

HepGentox: a novel promising HepG2 reportergene-assay for the detection of genotoxic substances in complex mixtures.

BackgroundIn risk assessment, genotoxicity is a key factor to determine the safety for the consumer. Most in vitro genotoxicity assays were developed for the assessment of pure substances. However, in recent years more attention has been given to complex mixtures, where usually low amounts of a substance are present. For high-throughput screening, a toxicologically sensitive assay should be used, covering a broad range of genotoxic substances and detecting them at low concentrations. HepG2 cells have been recommended as one of the prime candidates for genotoxicity testing, as they are p53 competent, less prone towards cytotoxic effects and tend to have some metabolic activity.MethodsA HepG2 liver cell line was characterized for its suitability for genotoxicity assessment. For this, a luciferase based reporter gene assay revolving around the p53 pathway was validated for the analysis of pure substances and of complex mixtures. Further, the cell’s capability to detect genotoxins correctly with and without an exogenous metabolizing system, namely rat liver S9, was assessed.ResultsThe assay proved to have a high toxicological sensitivity (87.5%) and specificity (94%). Further, the endogenous metabolizing system of the HepG2 cells was able to detect some genotoxins, which are known to depend on an enzymatic system. When complex mixtures were added this did not lead to any adverse effects concerning the assays performance and cytotoxicity was not an issue.DiscussionThe HepGentox proved to have a high toxicological sensitivity and specificity for the tested substances, with similar or even lower lowest effective concentration (LEC) values, compared to other regulatory mammalian assays. This combines some important aspects in one test system, while also being less time and material consuming and covering several genotoxicity endpoints. As the assay performs well with and without an exogenous metabolizing system, no animal liver fractions have to be used, which application is discussed controversially and is considered to be expensive and laborious in sample testing. Because of this, the HepGentox is suitable for a cost-efficient first screening approach to obtain important information with human cells for further approaches, with a relatively fast and easy method. Therefore, the HepGentox is a promising assay to detect genotoxic substances correctly in complex mixtures even at low concentrations, with the potential for a high throughput application. In a nutshell, as part of an in vitro bioassay test battery, this assay could provide valuable information for complex mixtures.

Time-Resolved Effect of Interferon-Alpha 2a on Activities of Nuclear Factor Kappa B, Pregnane X Receptor and on Drug Disposition Genes.

Interferon-alpha (IFN-α) is suggested to cause pharmacokinetic drug interactions by lowering expression of drug disposition genes through affecting the activities of nuclear factor kappa B (NF-ĸB) and pregnane X receptor (PXR). The time-resolved impact of IFN-α 2a (1000 U/mL; 5000 U/mL; 2 h to 30 h) on the activities of NF-ĸB and PXR and mRNA expression (5000 U/mL; 24 h, 48 h) of selected drug disposition genes and on cytochrome P450 (CYP3A4) activity in LS180 cells (5000 U/mL; 24 h, 48 h) was evaluated using luciferase-based reporter gene assays, reverse transcription polymerase chain reaction, and luminescence-based CYP3A4 activity assays. The cross-talk between NF-ĸB activation and PXR suppression was evaluated by NF-ĸB blockage (10 µM parthenolide). IFN-α 2a initially (2 h, 6 h) enhanced NF-ĸB activity 2-fold and suppressed PXR activity by 30%. mRNA of CYP3A4 was halved, whereas UGT1A1 was increased (1.35-fold) after 24 h. After 48 h, ABCB1 expression was increased (1.76-fold). CYP3A4 activity remained unchanged after 24 h, but was enhanced after 48 h (1.35-fold). IFN-α 2a demonstrated short-term suppressive effects on PXR activity and CYP3A4 mRNA expression, likely mediated by activated NF-ĸB. Longer exposure enhanced CYP3A4 activity. Clinical trials should evaluate the relevance by investigating the temporal effects of IFN-α on CYP3A4 using a sensitive marker substrate.

In vitro evidence suggesting that the toll-like receptor 7 and 8 agonist resiquimod (R-848) unlikely affects drug levels of co-administered compounds

Resiquimod (R-848) is an immune response modifier activating toll-like receptor 7 and 8. Its potential to cause pharmacokinetic interactions with concurrently administered drugs is unknown.To study the time course of the effect of resiquimod in LS180 cells as a model for intestinal tissue, luciferase-based reporter gene assays and reverse transcription polymerase chain reaction were used to investigate whether resiquimod affects the activities of nuclear factor kappa B (NF-ĸB), pregnane x receptor (PXR) or the transcription of selected central genes for drug disposition (cytochrome P-450 isozyme 3A4 (CYP3A4), CYP1A1, UDP-glucuronosyltransferase 1A1 (UGT1A1), ATP-binding cassette transporters ABCC2, ABCB1). Its impact on the activities of organic anion transporting polypeptides 1 or 3 (OATP1B1/3), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) or CYP3A4 was evaluated using fluorescence- or luminescence-based activity assays.Resiquimod irrelevantly increased NF-ĸB activity after 2 h (1 µM: 1.07-fold, P = 0.0188; 10 µM: 1.09-fold, P = 0.0142), and diminished it after 24 h (1 µM: 0.64-fold, P < 0.0001; 10 µM: 0.68-fold, P < 0.0001) and 30 h (10 µM: 0.68-fold, P = 0.0003). Concurrently, PXR activity after 24 h was marginally increased by 10 µM (1.05-fold, P = 0.0019). Resiquimod did not alter mRNA expression levels, activities of uptake or efflux transporters, or CYP3A4 activity.Given the marginal effects on NF-ĸB, PXR, expression levels of selected PXR target genes, and activities of important drug transporters and CYP3A4 in vitro, resiquimod is not expected to cause major pharmacokinetic drug-drug interactions in vivo.

Activation of human nuclear receptors by perfluoroalkylated substances (PFAS)

Perfluoralkylated substances (PFAS) such as perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS) are used to produce, e.g., surface coatings with water- and dirt-repellent properties. These substances have been shown to be hepatotoxic in rodents, and the mechanism of action is mostly attributed to the PFAS-mediated activation of the peroxisome proliferator-activated receptor alpha (PPARα). In the present study, we investigated by using luciferase-based reporter gene assays whether PFOA, PFOS and six alternative PFAS can activate, in addition to PPARα, eight other human nuclear receptors. All tested PFAS except for perfluorobutanesulfonic acid (PFBS) were able to activate human PPARα. Perfluoro-2-methyl-3-oxahexanoic acid (PMOH) and 3H-perfluoro-3-[(3-methoxypropoxy) propanoic acid] (PMPP) were weak agonists of human PPARγ. The other human nuclear receptors (PPARδ, CAR, PXR, FXR, LXRα, RXRα and RARα) were not affected by any PFAS tested in this study. Although PMOH was more effective than PFOA in stimulating PPARα in the transactivation assay, it was less effective in stimulating PPARα-dependent target gene expression in human HepG2 hepatocarcinoma cells. Notably, any effect observed in this in vitro study only occurred at concentrations higher than 10 μM of the respective PFAS which is in all cases several magnitudes above the average blood concentration in the Western population. Thus, the results suggest that nuclear receptor activation may only play a minor role in potential PFAS-mediated adverse effects in humans.

P14.52 Differential methylation of a single CpG site in the CD95 ligand promoter affects gene activity and correlates with invasiveness of glioma cells

Abstract BACKGROUND CD95 ligand (TNFSF6/APO-1L/FASLG) is a member of the Tumour Necrosis Factor Super Family (TNF-SF). Binding of the CD95 ligand (CD95L) to CD95 expressed on intrinsically apoptosis-resistant cancer cells like glioblastoma, triggers intracellular signal transduction resulting in increased tumour growth and invasiveness. A phase II clinical trial in patients with recurrent glioblastoma (NCT01071837) treated by Asunercept (a recombinant glycosylated fusion protein, which selectively binds and inhibits CD95L activity) was followed by a genome-wide assessment of DNA methylation. Here, a CpG-site (designated CpG2) within the CD95L-promotor was identified, exhibiting differential methylation between Asunercept responders (PFS&gt;5 months) and non-responders (PFS &lt; 2 months) with a significant survival benefit achieved in Asunercept treated patients with low CpG2 methylation (HR 0.34, p = 0.025). METHODS AND RESULTS We have performed in vitro studies to establish a link between the CpG2 methylation status and cellular characteristics of glioblastoma cell lines. A 3-dimensional spheroid invasion assay showed that highly methylated glioma cells like T98G did not grow and invade the surrounding matrix as aggressively compared to spheroids formed from low methylated glioma cells, e.g. U87-MG. Invasive growth of U87-MG spheroids in these assays was suppressed in the presence of Asunercept. A 1 kb fragment of the T98G CD95L promoter was subsequently cloned into a CpG-free reporter gene plasmid. Luciferase-based reporter gene assays of in vitro methylated and unmethylated plasmids in transfected HEK cells indicate that the CD95L promoter, despite the observed sparseness of CpG sites, is at least partially regulated by its methylation level. Furthermore, mutational disruption of the CpG2 site completely silenced reporter gene activity in vitro, which insinuates that both methylation level and gene promoter sequence are involved in regulation of the CD95L gene. CONCLUSION In essence, the level of CpG2 methylation correlates to aggressiveness of glioma derived cancer spheroids in vitro, and methylation of the CD95L promoter in glioblastoma tissue from patients might warrant use as a potential biomarker predicting response to therapy with Asunercept. We are currently developing a specific and sensitive assay to quantify CpG2 methylation considered as a companion diagnostic for further clinical studies.

Hypermethylation of the serotonin transporter gene promoter in panic disorder–Epigenetic imprint of comorbid depression?

Panic disorder (PD) is frequently comorbid with major depressive disorder (MDD), which has been associated with impaired treatment response and recovery rates. Alterations in the serotonergic system may play a crucial role in the pathogenesis of PD and MDD and might constitute a shared biological trunk of both disorders. Epigenetic patterns such as hypermethylation of the serotonin transporter gene (SLC6A4) have been associated with various mental disorders including MDD, but, to date, no association with PD has been reported. In the present study, SLC6A4 promoter methylation was investigated in two independent samples of PD patients in a case-control design (sample 1: N = 120; sample 2: N = 118), while – given the reported high comorbidity of both disorders – taking into account the effect of comorbid MDD. The functional relevance of altered SLC6A4 promoter methylation was investigated by means of luciferase-based reporter gene assays. SLC6A4 promoter hypermethylation in PD patients relative to healthy controls was driven by comorbid diagnosis of MDD (p = 9 × 10−6), whereas no altered methylation levels were observed in patients without comorbid MDD. This held true not only in comparison to healthy controls, but also in direct comparison between PD patients with and without comorbid MDD (p = .009). Functional analyses revealed increased methylation of the investigated region to confer decreased reporter gene activity. The present results suggest functionally relevant SLC6A4 promoter hypermethylation as a possibly specific epigenetic marker of MDD, but not of PD itself, and thus might constitute a selective biomarker informing differential diagnosis based on individual epigenetic profiles.

MiR-25 Promotes Melanoma Progression by regulating RNA binding motif protein 47.

Melanoma is the most aggressive skin cancer, and accounts for the major part of skin cancer-related deaths in the world. In addition, the underlying mechanism of tumor progression in melanoma remains far from being elucidated. In this study, we have evaluated the function of miR-25 in melanoma. First, we examined the expression of miR-25 in four melanoma cell lines (A875, MV3, M14 and uacc-257) and in a normal melanocyte cell line (HEM-a). Then, we overexpressed miR-25 in M14 cells. Our results show that miR-25 promotes M14 cell proliferation and migration. We found that miR-25 up-regulates the PI3K/Akt/mTOR signaling pathway in these tumor cells. Furthermore, a luciferase-based reporter gene assay showed that miR-25 could directly target the RNA-binding motif protein 47 (RBM47). Taken together, our findings suggest that RBM47 is a promising target for the treatment of melanoma.

Plasticity of Functional MAOA Gene Methylation in Acrophobia.

Epigenetic mechanisms have been proposed to mediate fear extinction in animal models. Here, MAOA methylation was analyzed via direct sequencing of sodium bisulfite-treated DNA extracted from blood cells before and after a 2-week exposure therapy in a sample of n = 28 female patients with acrophobia as well as in n = 28 matched healthy female controls. Clinical response was measured using the Acrophobia Questionnaire and the Attitude Towards Heights Questionnaire. The functional relevance of altered MAOA methylation was investigated by luciferase-based reporter gene assays. MAOA methylation was found to be significantly decreased in patients with acrophobia compared with healthy controls. Furthermore, MAOA methylation levels were shown to significantly increase after treatment and correlate with treatment response as reflected by decreasing Acrophobia Questionnaire/Attitude Towards Heights Questionnaire scores. Functional analyses revealed decreased reporter gene activity in presence of methylated compared with unmethylated pCpGfree_MAOA reporter gene vector constructs. The present proof-of-concept psychotherapy-epigenetic study for the first time suggests functional MAOA methylation changes as a potential epigenetic correlate of treatment response in acrophobia and fosters further investigation into the notion of epigenetic mechanisms underlying fear extinction.

Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine.

The two endonucleases XPF and XPG are essentially involved in nucleotide excision repair (NER) and interstrand crosslink (ICL) repair. Defects in these two proteins result in severe diseases like xeroderma pigmentosum (XP). We applied our newly CRISPR/Cas9 generated human XPF knockout cell line with complete loss of XPF and primary fibroblasts from an XP-G patient (XP20BE) to analyze until now uncharacterized spontaneous mRNA splice variants of these two endonucleases. Functional analyses of these variants were performed using luciferase-based reporter gene assays. Two XPF and XPG splice variants with residual repair capabilities in NER, as well as ICL repair could be identified. Almost all variants are severely C-terminally truncated and lack important protein-protein interaction domains. Interestingly, XPF-202, differing to XPF-003 in the first 12 amino acids only, had no repair capability at all, suggesting an important role of this region during DNA repair, potentially concerning protein-protein interaction. We also identified splice variants of XPF and XPG exerting inhibitory effects on NER. Moreover, we showed that the XPF and XPG splice variants presented with different inter-individual expression patterns in healthy donors, as well as in various tissues. With regard to their residual repair capability and dominant-negative effects, functionally relevant spontaneous XPF and XPG splice variants present promising prognostic marker candidates for individual cancer risk, disease outcome, or therapeutic success. This merits further investigations, large association studies, and translational research within clinical trials in the future.

Impact of enzalutamide and its main metabolite N‐desmethyl enzalutamide on pharmacokinetically important drug metabolizing enzymes and drug transporters

Enzalutamide is a new drug against castration-resistant prostate cancer. Recent data indicate profound induction of drug metabolizing enzymes (e.g. cytochrome P450 isoenzyme (CYP) 3A4) but comprehensive in vitro data on other CYP enzymes, drug conjugating enzymes or drug transporters is scarce. Moreover, the mechanisms of induction are poorly investigated and the effects of the active metabolite N-desmethyl enzalutamide are unknown. Using LS180 cells as an induction model and quantitative real-time reverse transcription polymerase chain reaction, our study demonstrated a concentration-dependent induction of CYP1A1, CYP1A2, CYP3A5, CYP3A4, UGT1A3, UGT1A9, ABCB1, ABCC2 and ABCG2 mRNA. Induction of CYP3A4 and ABCB1 was confirmed by Western blot analysis and is likely mediated by activation of the nuclear receptor pregnane x receptor, elucidated by a luciferase-based reporter gene assay. Enzalutamide's main active metabolite N-desmethyl enzalutamide exhibited only weak induction properties. mRNA expression of UGT2B7 was suppressed by enzalutamide and its metabolite. Both compounds are apparently not transported by P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP). N-desmethyl enzalutamide more potently inhibited important drug transporters (P-gp, BCRP, OATPs) than enzalutamide. Taken together, the pharmacokinetics of concurrently administered drugs is likely altered during enzalutamide therapy. Levels of metabolically (mainly CYP3A4) eliminated drugs are expected to be decreased, whereas the abundance of compounds with solely transporter-determined pharmacokinetics (P-gp, OATPs) is likely enhanced.

Assessment of novel tobacco heating product THP1.0. Part 6: A comparative in vitro study using contemporary screening approaches

Cigarette smoking is a major risk factor for many adverse health conditions. Novel tobacco heating products (THPs) heat tobacco, reducing exposure to many of the harmful combustion toxicants in conventional cigarette emissions. In vitro studies have been employed to support the toxicological evaluation of chemicals and complex mixtures, including cigarette smoke. The use of automated robotics platforms for in vitro toxicological screening complements traditional testing approaches. Multiparametric toxicity and oxidative stress endpoints were used to assess in vitro biological responses elicited after exposure to total particulate matter (TPM) from two commercially available THPs, and the reference tobacco product 3R4F, in human bronchial epithelial cells. A luciferase-based reporter gene assay was used to assess antioxidant response element (ARE) transcriptional activation in stably transfected H292 cells after 6 and 24 h exposures. High-content screening was used to assess 10 endpoints normal human bronchial epithelial cells after 4 or 24 h exposures. 3R4F TPM stimulated significant increases in ARE activation (p < 0.005) and moderate activity in HCS cell-based assays compared to THP at comparable doses. THPs showed little or no activity in all assays. HCS techniques can extend safety assessments providing information quickly in the early stages of product innovation and development.

CRHR1 promoter hypomethylation: An epigenetic readout of panic disorder?

The corticotropin releasing hormone receptor 1 (CRHR1) is crucially involved in the hypothalamic-pituitary-adrenal axis and thus a major regulator of the stress response. CRHR1 gene variation is associated with several mental disorders including anxiety disorders. Studies in rodents have demonstrated epigenetic regulation of CRHR1 gene expression to moderate response to stressful environment. In the present study, we investigated CRHR1 promoter methylation for the first time regarding its role in panic disorder applying a case-control approach (N=131 patients, N=131 controls). In an independent sample of healthy volunteers (N=255), CRHR1 methylation was additionally analyzed for association with the Beck Anxiety Inventory (BAI) score as a dimensional panic-related intermediate phenotype. The functional relevance of altered CRHR1 promoter methylation was investigated by means of luciferase-based reporter gene assays. In panic disorder patients, a significantly decreased CRHR1 methylation was discerned (p<0.001). Accordingly, healthy controls with high BAI scores showed significantly decreased CRHR1 methylation. Functional analyses revealed an increased gene expression in presence of unmethylated as compared to methylated pCpGl_CRHR1 reporter gene vectors. The present study identified a potential role of CRHR1 hypomethylation – conferring increased CRHR1 expression – in panic disorder and a related dimensional intermediate phenotype. This up-regulation of CRHR1 gene expression driven by de-methylation might constitute a link between the stress response and panic disorder risk.

NDST1 Preferred Promoter Confirmation and Identification of Corresponding Transcriptional Inhibitors as Substrate Reduction Agents for Multiple Mucopolysaccharidosis Disorders.

The stepwise degradation of glycosaminoglycans (GAGs) is accomplished by twelve lysosomal enzymes. Deficiency in any of these enzymes will result in the accumulation of the intermediate substrates on the pathway to the complete turnover of GAGs. The accumulation of these undegraded substrates in almost any tissue is a hallmark of all Mucopolysaccharidoses (MPS). Present therapeutics based on enzyme replacement therapy and bone marrow transplantation have low effectiveness for the treatment of MPS with neurological complications since enzymes used in these therapies are unable to cross the blood brain barrier. Small molecule-based approaches are more promising in addressing neurological manifestations. In this report we identify a target for developing a substrate reduction therapy (SRT) for six MPS resulting from the abnormal degradation of heparan sulfate (HS). Using the minimal promoter of NDST1, one of the first modifying enzymes of HS precursors, we established a luciferase based reporter gene assay capable of identifying small molecules that could potentially reduce HS maturation and therefore lessen HS accumulation in certain MPS. From the screen of 1,200 compounds comprising the Prestwick Chemical library we identified SAHA, a histone deacetylase inhibitor, as the drug that produced the highest inhibitory effects in the reporter assay. More importantly SAHA treated fibroblasts expressed lower levels of endogenous NDST1 and accumulated less 35S GAGs in patient cells. Thus, by using our simple reporter gene assay we have demonstrated that by inhibiting the transcription of NDST1 with small molecules, identified by high throughput screening, we can also reduce the level of sulfated HS substrate in MPS patient cells, potentially leading to SRT.

Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays

Firefly luciferase-based reporter gene assays are the most commonly used assays to investigate the transcriptional regulation of gene expression. However, direct interaction of tested compounds with the firefly luciferase leading to altered enzymatic activity may lead to misinterpretation of experimental data. When investigating the proteasome inhibitors bortezomib, carfilzomib, and ixazomib, we observed increased luminescence for bortezomib and ixazomib, but not for carfilzomib, in a pregnane-X-receptor (PXR) reporter gene assay, which was inconsistent with the mRNA expression levels of the main PXR target gene CYP3A4. To further scrutinize this phenomenon, we performed experiments with constitutively expressed firefly luciferase and demonstrated that the increase in cellular firefly luciferase activity is independent from PXR activation or CYP3A4 promoter. Using cell-free assays with recombinant firefly luciferase enzyme, we made the counterintuitive observation that firefly luciferase activity is inhibited by bortezomib and ixazomib in a reversible and competitive manner. This inhibition stabilizes the firefly luciferase enzyme against proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life with subsequent enhancement of total cellular luminescence that eventually mimicked PXR-driven luciferase induction. These data show that particular compounds can strikingly interfere with firefly luciferase and once more illustrate the importance of careful interpretation of data obtained from luciferase-based assays.